Immunological reagent and radioimmuno assay

ABSTRACT

A broad new class of reagents permits extremely sensitive and specific assay for, or chemical separation of, a broad range of biological and nonbiological substances. Each reagent consists of a suspension of microscopic carrier material particles, each particle bearing (1) tracer material-fluorescent, radioactive or otherwise - and (2) a coating of biological antibody for the substance whose assay is desired. The latter substance if introduced into the suspension links the particles together in pairs or clumps, which may be sensitively and accurately detected by monitoring the tracer. The carrier is preferably partially hydrolyzed polyacrylamide resin, or in appropriate applications acrylic acid and other derivatives thereof, and other polymers including agar, and the coupling effected by covalent bonding. Other embodiments, including various mechanical forms of carrier, for greater ease of handling and separation, are also described.

il'nited States Patent [191 @reyer 1 Dec. 10, 1974 1 HMMUNOLOGICALREAGENT AND RADIOIMMUNO ASSAY Inventor: William J. Dreyer, 2369Highland, Altadena, Calif. 91001 Filed: Sept. 1, 1971 Appl. No.: 177,017

U.S. Cl. 424/1, 23/230 B, 250/303, 424/8, 424/12 Int. Cl. A61k 27/04,GOln 31/22 Field of Search 424/1, 8, 12; 23/230 B; 252/30l.l R; 250/303References Cited UNITED STATES- PATENTS 2/1966 Pollack 424/12 4/1970Axen et a1. 424/12 1/1971 Axen ct a1. 424/1 7/1971 Wolf 424/1 2/1972Catt 424/1 UX OTHER PUBLICATlONS Aorameas et al., The Journal ofBiological Chem, [Vol 242, No. 7, Issue of April 10, pp. 1651-1659],(1967), [OP 501JT].

Primary ExaminerBenjamin R. Padgett Attorney, Agent, or Firm-William W.Haefliger 5 7] ABSTRACT A broad new class of reagents permits extremelysensitive and specific assay for, or chemical separation of, a broadrange of biological and nonbiological substances. Each reagent consistsof a suspension of microscopic carrier material particles, each particlebearing (1) tracer materia1fluorescent, radioactive or otherwise and (2)a coating of biological antibody for the substance whose assay isdesired.

8 Claims, 1 Drawing Figure IMMUNOLOGICAL REAGENT AND RADIOIMMUNO ASSAYBACKGROUND OF THE INVENTION The present invention is directed to theconcept that the immune reaction and its products can be used forchemical separations and assays. This concept is, for example,implemented by causing an organism (often a mammal, such as a rabbit,mouse or horse) to generate antibody to a particular substance-asubstance which is to be the object of an assay or separation.

The antibody thus formed may be used to seek further quantities of theparticular substance which produced it, and the resulting reactions maybe used in a vast variety of ways-to precipitate or agglutinate theparticular substance out of solution or suspension, thus indicating itspresence qualitatively or quantitatively while effecting a separation;or to couple the particular substance to tracer material, or solid orsemi-solid material, pre-attached to the antibody.

Some of these applications have been known for years but are greatlyfacilitated, and rendered amenable to automation, by the presentinvention; others of these applications have been conceived of only inthe light of the present invention, and would not have been at allpractical or meaningful previously. To suggest the enormous power of theinstant invention, there are listed below a number of its applications.

This listing represents only a few of the potentialities of immune assayand separation, which are unrealizable or imperfectly realizable withoutthe present invention:

1. location, separation and measurement of cholesterol, hormones,viruses and their antigens, or antivirus antibodies, in serum;

2. quantitative assay for enzymes by their presence-not merely by anindirect measure of their activity;

3. assessment of the character and/or effectiveness of an organismsimmune-system response, or of its suppression-as, for example,preparatory to transplant surgery; and

4. assay for an almost unlimited variety of miscellaneoussubstancesincluding even omnipresent biological materials such assteroids-by using techniques involving attachment of suchlowmolecular-weight molecules to proteins.

Not only research analyses and separations, but clinical and otherroutine uses are contemplated.

It is felt that great advances in medical science and practice hinge onthe application of the immune mechanisms to these listed areas andothers; and that such application in turn hinges on the presentinvention, which at once renders immunological separations and assayssignificantly more sensitive, reproducible, accurate, convenient andamenable to automation than heretofore possible.

Radioactive, fluorescent and other tracers or tags" are used extensivelyin biochemistry, and certainly immunology is not an exception. However,in the context of the present invention they are used in a way which isbelieved novel and unobvious.

Conventionally, tracers are coupled directly to one of the molecules orcells of interest in a particular reaction or assay; sometimes tracersare coupled via intermediaries which simply serve to supply appropriatechemical combining properties for both the active component and thetracer.

Radioactive tracers have the disadvantage that their radioactive decaycannot be turned off or on at will. Thus if high activity, to attainhigh sensitivity, is de sired, the tagged reagent is continuallybombarded from the time of its manufacture, with ionizing anddestructive radiation. Not only does the radioactivity itself havelimited life, but the reagent steadily deteriorates, further limitingthe useful life and increasing the probability of nonspecific reactions.Radioactive tracers may therefore be quite unusable, for applications ofthe sort considered here, in remote locations.

In addition, as is well known, radioactive substances are dangerous.Persons who handle them are required by common sense and by law to havespecial training. Furthermore their detection requires very expensive,special purpose instrumentation.

They give high sensitivity (though not nearly so high as certainfluorescent label molecules), and while widely used for this reasontheir applications have been limited mainly to well-equipped researchlaboratories.

Fluorescent tracers yield overall reaction-detection sensitivitieswhich, though a great deal better than those obtainable with radioactiveand other sorts of tags, have still heretofore left much to be desired.Nevertheless, tracers have been used extensively for localization ofspecific constituents at the outer surfaces of cells, or withincells-that is, in cytology. Fluorescent tracer work with immunereactants, generally known as immune-fluorescence, has heretofore beenput to use primarily in cytology. Since cells are in a sense particles,the distinction between immuno-fluorescence and parts of the presentinvention is essential. In immuno-fluorescence the biological cells(particles) are themselves the objects of a screening survey; while inthe present invention the particles (or other carrier forms) are part ofa chemical tool used for assays and separations of other substances, asexplained later.

Fluorescent tracers have also been used in other immunological work-butin such other instances the tracer molecules have simply been added orattached to immune reagent molecules on a roughly one-to-one basis.

Next, the use of coupled particles is discussed.

Immune reactions classically involved mechanical effects-precipitationor agglutination-and so it was natural to attempt to provide mechanicalamplification for these phenomena. Experimenters since the early 1940 shave employed techniques involving attachment of immune reactants toparticulate or extended solids, carriers. With partial success,particulate solids have been used both in suspension and packed invarious kinds of flow-through columns; the reactant which is originallynot attached to the solid is then linked to the solid by reaction withthe pre-attached homologue. (An antibody is the immunological homologueof the antigen which produced it, and vice versa.)

This produces various effects, depending on the size range of solidinvolved: for tiny particles of carrier, the reactant couples togetherlarge masses of particles; for extended carrier material, the reactantsimply accumulates at the spatially-fixed location of the carrier; forspatially semi-fixed particles packed in flow-through columns, theoriginally-unattached reactant accumulates in the column (actinggenerally as if the packed particles were a porous extended carrier).

In this way, through a simple mechanical amplification of the observableeffects of immune reaction, workers in immunology have been able toelucidate many important aspects of the immune reaction.

In another approach to amplifying the observable effects of immunereaction, J. H. Brewer (US. Pat. No. 3,074,853) and Robert W. Terry (US.Pat. No. 2,301,717) have described techniques for coprecipitation ofsuspended pigment particles with immunoprecipitates, providing a helpfulcolor advantage in the observation of immune reactants-this techniquebeing limited (1) to work in which a precipitate is actually formed, andbeing further limited in that (2) the quantity of pigment precipitatedis not reliably quantitatively related to the extent of agglomeration,and (3) the pigment will coprecipitate with, generally, any precipitatewhich forms, being almost completely nonspecific to the reaction ofinterest.

Robert W. Terry has also reported (US. Pat. No. 2,194,131) the use ofstained antigen in assaying for antibody; his antigen happened to beSalmonella organisms.

In all of these mechanical-amplification and related schemes, and inTerrys stained-antigen technique, observation remains relatively clumsy,and the techniques remain with few exceptions the exclusive property ofthe careful academic worker.

The reason for this failure of the immunological amplified clumpingapproach to become a dominant analytical technique in biochemistry andmedicine is clear: mere mechanical amplification is inadequate for manyapplications.

SUMMARY OF THE INVENTION In accordance with the present invention,immune reactants are coupled, preferably by covalent bonding, to carriermaterial. For assay purposes the carrier material has associated with ita sizable quantity of tracer material, such as fluorescent tracer.

In one preferred embodiment, the carrier is in the form of microscopicparticles in a fluid suspension. In other embodiments the carriermaterial may be used in macro-reticular form-that is, in the form of amicroscopic particle or strand having a convoluted, open internalstructure, and thus presenting extremely high surface area, per unitvolume, for covalent attachment of reactants.

The carrier materials may be made to accept attachment of largequantities of reactants and, more importantly, large quantities oftracer material-so that the various difficulties mentioned in thepreceding pages are obviated, as explained herebelow.

As noted in the prior art portion of this specification, immune-reactantattachment to particulate or other carrier material provides a means oflinkage between the carrier material and the homologue of the attachedreactant. Particulate carrier material is thus subject to clumping orprecipitation, with extremely high specificity, as homologue moleculeslink two or more reactant-coated particles. The extent of this specificagglutination or precipitation then serves as a measure of the amount ofhomologue present. As the particles are in general much larger than thereactant molecules, detection of carrier-particle clumping is easierthan direct detection of reactant clumping.

But this sort of mechanical amplification is limited in its ability torender assays sensitive and amenable to automation.

The benefits of the present invention transcend mere mechanicalamplification, for the large quantities of tracer which may be borne bythe carrier material permit increased sensitivity and thus completelyautomated, quantitative, photoelectric measurement and monitoring.

BRIEF DESCRIPTION OF THE DRAWING Greater understanding of the characterand benefits of my invention may be had through reference to thefollowing description of embodiments, and to the accompanying singledrawing, which is a highly schematic representation on a microscopicscale, of certain physical relationships related to the practice of theinvention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS In the accompanying FIGURE,carrier particles It) and are represented as suspended in a solution.Tracer molecules symbolized as T, and also identitied at 12 and 112typically, are associated with the carrier particlespreferably, but notnecessarily, by distribution generally throughout the volume of eachparticle, as indicated.

Molecules or other units of homologue to a substance whose assay issought are attached, preferably by covalent chemical bonding, to thesurfaces of the particles 10 and 110. The homologue units are symbolizedas H, and also are identified typically at 11 and 1111.

Also suspended, or dissolved, in the solution are molecules or otherunits of the substance whose assay is sought, the reactant under assay,which is symbolized as -R- and typically identified at 13. Some units ofthis reactant are typically attached to one or another of the homologueunits, as indicated typically at 114 and 1114. As particles having suchattachments drift about in the solution, reactant units -R-statistically form linkages between two particles, as indicated byreactant at 113 linking together particles 110. Depending uponconcentrations and other conditions, the most favored linkage conditionmay be pairs as illustrated, or larger pluralities or multiplicities byattachment of further particles such as 110 to reactant at 114, or byattachment of reactant on further particles 10, as indicated at 14, tohomologue 111 on already agglomerated particles 110.

In either the pair or higher-agglomerate case, the degree ofagglomeration is readily ascertained through detection of thecorresponding degree of agglomeration of tracer T, the pair or higheragglomerate providing a larger signal pulse from correspondinglyagglomerated tracer 1112 than provided by theindividual-particle-carried tracer 112.

The accompanying FIGURE indicates the particles 10 and 110 as generallyconvex everywhere about their surfaces, but the particle surface neednot be so. In fact, for some applications a highly convoluted surfacestructure or a macro-reticular form having partially closed encagingstructure, thereby permitting attachment or encagement of largequantities of homologue, is preferable; the preferred structure in otherapplications is a strand, of microscopic cross-section; the use of suchstructure is within the scope of the present invention.

The many applications in serology, and particularly human serology, arefurther enhanced by the low tendency of the acrylamides towardnon-specific reaction, and other favorable properties of theacrylamides. However, many other carrier materials, such as otheracrylic acid derivatives, agar, Agarose, and other resins may be used tovarying degrees of advantage within the scope of the invention.

In the earliest immunological experiments, antibody was used in clumpingof bacteria, viruses and cells; red blood cells were used as carriers insome of these experiments. These biological units soon provedinconvenient for assay work, being cumbersome to mantain in usablecondition and to handle, and led Paul R. Cannon and Charles E. Immunol.(J. Immunol 38 p. 365, 1940) to apply synthetic carrier particles-tinyglobules of collodion, which is cellulose acetate or nitrate, toimmunological assays.

The collodion was little better, having a strong tendency towardunstablenon-specific reaction or adsorptive binding with the immune reactants orother substances present.

Many workers have tried other carriers to which immune reactants wereattached by surface adsorption. The problems here were well expressed in1963 by A. T. Jagendorf et al. (Biochim. Biophys. Acta 78, p. 516) andA. H. Sehon (Brit. Med. Bull. 19, p. 183). The latter writer discussesthe use of covalent bonding between antigens and carrier material, as animprovement relative to the adsorptive attachment.

However, even covalent bonding per se did not completely solve theproblems, because of deficiencies of the then-used carrier materialsthemselves. This will now be pointed up with reference to the currentlymost widely used and commercially successful immunoassay.

In the 1950s there was brought to light the concept of the so-calledlatex test, now well-known in clinical work as a test for pregnancy, andin certain other assays for hormones.

The carrier particles here are a latex (that is, a suspension) ofpolystyrene. A system for producing polystyrene particles for thispurpose has been patented by R. T. Fisk (US. Pat. No. 3,088,875), theclaims being directed to a particular range of particle sizes. While thelatex test is extremely widespread in use, it is so only because nothingbetter has until recently been available: the mechanical problems areonly partially removed.

The latex particles have a tendency to stick to each other and tocontainers; their preparation, moreover, involves development ofconsiderable special technique-being far from straightforward.

Their chemical properties are further aggravations. Immune reactants aregenerally coupled to the polystyrene by hydrophobic bondingthat is,attachment via molecular groups on the polystyrene whose affinity forwater is lower than their affinity for groupings on the immunereactants-which is a very weak form of attachment, having the samegeneral strength as immunological affinities; consequently immunereactants may be stripped away from the carrier upon reaction with theirhomologues. Also, techniques for attachment of many immune reactants tothe latex are not available.

Finally, the latex has a variable, relatively high tendency towardnon-specific attachment. This means that in tests where it can be usedat all, the results of the test must be assessed statistically, becausea significant (variable) amount of agglutination is almost alwaysobserved even if no homologue is present in the test solutron.

In the latex pregnancy test, for example, the clumping observed in asolution derived from the patients urine must be carefully comparedvisually with the clumping in a standard solution, and the relativeamount of agglutination must be given a numerical rating by the visualobserver. Apart from the obviously objectionable subjectivity of thismethod, the important fact stands out that the immunologicalnonspecificity and variability of the latex prevents a simple yes-or-noanswer to the question Is the patient pregnant?" Instead the answer mustbe given in probabilistic relative terms--while there is nothingprobabilistic or relative about the question. The test is in fact ofteninconclusive.

Thus the polystyrene particles, hydrophobically bonded to immunereactants, are relatively unsatisfactory in preparation, in use, and inmeasurement significance.

In related work, R. R. Porter et al. (Ann. Rev. Biochem. 31 p. 625,1962) and Silman and Katchalski (Ann. Rev. Biochem. 35, p. 873, 1966)have reported problems with antigen covalently coupled to diazoaminopolystyrene, and with poly-p-aminostyrene, respectively.

Nevertheless, the latex test, with all its drawbacks, incorporates oneof the most advanced combinations of materials and bonding chemistryheretofore known in immunological applications.

In more recent work, much less widely practiced,

' acrylic acid derivatives have been used as immunocarriers (Manecke etal., Pure Appl. Chem. 4 p. 507, 1962), and in another approach to thecarrier tech nique some investigators have encaged immune reactantswithin closed structures-generally microscopic particles (Silman andKatchalski, Ann. Rev. Biochem. 35, p. 873, 1966; and Goodfriend et al.,Immurwchem. 6, p. 481, 1969). Inman and Dintzis (Biochem. 8, p. 4074,1969) have described acrylamide carrier chemistry which is particularlywell suited for use with the present invention.

The acrylic acid and acrylamide techniques are highly preferred to theothers discussed hereabove, but the instant invention is not limited touse with these particular chemicals, others such as the polystyrenelatex, other covalentlybonded carrier materials, even collodion orbiological cells, being usable with the present invention-within thelimitations upon their usefulness stated herein.

The acrylamide resins are advantageous both for physical and chemicalproperties: they can be formed into a variety of shapes of varioussizes, such as microscopic globules or flecks, strands of microscopiccrosssection, or thin sheets; after formation these bodies are notmutually adherent nor prone to protein adsorption; and through partialhydrolysis or hydrazinolysis they can be made amenable to covalentbonding, which is stronger than the antigen-antibody affinities andconsequently provides stable attachment of carrier to antigen orantibody throughout the duration of reactions. No work is reportedheretofore using these materials with associated tracer molecules, andcovalently bonded to reactive substances in immunology.

Through the use of acrylamide resinor, where loose surface convolutionsare desirable, through the use of other polymers of acrylic acid or itsderivatives-the particles or other structures may be made exceedinglystable, and non-mutually cohesive. They are moreover, due to theiramenability to covalent bonding of immune reactants, capable of beingattached to such reactants in a manner which is much stronger thanimmunological bonding-and thus permanent with respect to the duration ofimmune reactions.

Finally, there appears a further advantage, with respect toimmunochemistry, of using the acrylamide resins: these carriersubstances, because of their molecular structure, are very little proneto nonspecific reaction with immune reactants. Acrylamide is amphoteric,hydrophilic and not mutually reactive (nor reactive with proteins thesebeing the three properties of proteins which render them relativelylittle subject to nonspecific reactions. These properties arecharacteristic of acrylic acids also, under appropriate conditions, butnot of polystyrene latex, collodion, or most other substances tried inthe past as carrier materials. Thus the acrylamide in particular makesit possible to greatly reduce interference with immune monitoring due tobackground" precipitation and agglutination, which are due in turn tonon-specific immunological attachments involving the carrier materialitself. Consequently the present invention is enhanced by the novelcombination of the favorable physical, chemical and immunologicalproperties of the acrylamide carrier and the covalent bond, thusrendering the present invention remarkably effective for manyapplications.

In the following paragraphs are listed various laboratory detailssuitable and helpful to practice of the present invention; it will beunderstood that other chemicals, materials and procedures may besubstituted for those presented here without departing from the scope ofthe instant invention as defined by the appended claims.

Particle Materials proteins 1. Acrylic acid-Rohm & Haas, type XE 256,suspension washed 3 times with 10 volumes of saline, resuspended to theoriginal volume in saline and stored at 4C.

2. Acrylamide-Calbiochem Bio-gel type P-150, 100 to 200 mesh. Nine gramssuspended in 350 ml of 2 N hydrochloric acid, allowed to hydrolyze atroom temperature (25C) with continuous shaking for 4 days, thenfiltered. By analysis of filtrate for ammonia (0.45 mg N/ml), 8.9% ofamide groupings were determined to have been hydrolyzed to carboxyl.Filtrate washed repeatedly with saline until neutral, resuspended in 150ml saline and stored cold.

3. Agarose-Calbiochem Bio-gel type A, 0.5 M, 100 to 200 mesh, suppliedin suspension and used as received.

Fluoroscent Dyes 1. F1uoresceinisothiocyanate-Calbiochem, catalog No.34321.

2. e-dansyl-L-lysine-Calbiochem A grade, catalog No. 251221.

Proteins l. Gamma-globulin (7s)Calbiochem human A grade, catalog No.345372, purified by dissolving to 1% in pH 8.4 buffered saline, andsubjected to chromatography on a column of Bio-gel P300, monitored by UVabsorption at 280 nm, collecting the second emerging peak of molecularweight about 160,000. This material concentrated to about 2.5% byultra-filtration and shell-frozen in small quantities in test tubes andstored frozen.

2. Bovine albuminpurified-by-crystallization grade, Sigma ChemicalCompany catalog No. A4378, further purified and stored in the same wayas the gamma globulin. (Molecular weight about 69,000.)

3. Antibody to human 7s gammaglobulinCalbiochem catalog No. 539817ten-fold purified rabbit antibody, A grade.

4. Antibody to bovine serum albumin-Calbiochem catalog No. 539807,ten-fold purified goat antibody, A grade. 1

Other Chemicals 1 water-solublecarbodiimide-1-ethyl-3-dimethylaminopropylcarbodiirnide, Ott ChemicalCo.

2. Hydrazine-Eastman Organic Chemicals, catalog No. 902.

3. Cyanogen bromide-Eastman Organic Chemicals, catalog No. 919.

4. Radioiodine, l sodium iodide, carrier-free, in neutral solution(available from Radiochemical Centre, Amersham, Bucks, England).

5. Chloramine-T-Eastman Organic Chemicals, catalog No. 1022.

6. EthylenediamineEastman Organic Chemicals, catalog No. 1915.

Radioactive Tagging of Acrylamide First react partially hydrolyzedacrylamide with tyrosine using carbodiimide activator, then iodinatethat compound with radioiodine by the method of W. M. Hunter and S. C.Greenwood (Nature 194, 495, 1962). The residual carboxyl groups of thetyrosine and of the hydrolyzed polyacrylamide can then be activated withcarbodiimide and coupled to the desired protein. The same proteincoupling procedure can be used with the dansyl-lysine-labelled acrylicacid or acrylamide, or the fluoresceinisothiocyanate-labelled acrylamide(see paragraph 3 under stained particles below). There are a number ofalternate procedures for coupling proteins to the various insoluble,synthetic polymer particles, as illustrated by the publication by Inmanand Dintzis, or as described in numerous textbooks. See, for example,Handbook of Experimental Immunology, edited by D. M. Weir, F. A. DavisCo, Philadelphia, 1967; or Methods in Immunology by D. H. Campbell eta1., W. A. Benjamin Inc., NY. 1964.

Stained Particles 1. Acrylic acid stained by e-dansyl-L-lysine-Dilutethe acrylic acid suspension 1:1 with saline, and adjust to pH 5.4 with 3N HCl. Add 10 ml to 1 ml 0.005 M e-dansyl-lysine and mg water-solublecarbodiimide. Store cold overnight, wash repeatedly with pH 9.4, 0.1 Mcarbonate-buffered saline until control prepared in the same way withoutcarbodiimide is completely non-fluorescent. Wash with pH 7.4 bufferedsaline until pH of supernatant is about 7.6.

2. Acrylamide stained with e-dansyl-lysine-To ml hydrolyzed acrylamidesuspension add 1 ml 0.005 M e-dansyl-lysine and 50 mg water-solublecarbodiimide. Adjust pH and maintain pH at 5.0 10.3 by continuoustitration with 0.5 N HCl at room temperature. After 2 hours, washrepeatedly, first with pH 9.5, 0.1 M carbonate-buffered saline, then inpH 7.4 phosphatebuffered saline, and resuspend to 5 ml in bufferedsaline. Substantially all of the dye is retained on the particles.

3. Acrylamide stained with fluoresceinisothiocyanate-To 1 ml ofhydrolyzed acrylamide suspension, in which the acrylamide has beencopolymerized with monomers containing amino groups, add 5 mgfluoresceinisothiocyanate dissolved in cold pH 9.4 carbonate-bufferedsaline, store cold overnight with continuous stirring, centrifuge, wash3 times with volumes of pH 9.3 carbonate-buffered saline, and 3 timeswith 10 volumes of pH 7.4 buffered saline. Substantially all of the dyeis retained on the particles.

4. Agarose coupled to dansyl lysine-Agarose may be activated withcyanogen bromide following the method of Cuatrecasas et al. (Proc. Nat.Acad. Sci. US 61 636, 1968). Add 5 ml of a suspension containing 3 ml ofactivated agarose in 0.1 molar carbonate buffer, pH 9.0, to 2 ml of thesame buffer containing 25 mg of e-dansyl-lysine, and hold 1 hour at 4C.Quickly filter this suspension in the cold, and wash with 5 volumes ofcold pH 9.0 buffer. Then immediately resuspend in 5 ml of a 56% solutionof the protein (antibody or antigen) to which it is to be coupled, andhold 24 hours more in the cold with stirring. Wash it twice with 10volumes of cold pH 9 buffer, then with cold pH 7.4 phosphate-bufferedsaline until pH falls to 7.6 or below. Resuspend the precipitate in 5 mlpH 7.4 saline and store cold.

Immunological Reactions Immunological reactions are carried out bymixing a few drops of a dilute suspension of the tagged andprotein-coated particles with a drop or two of serum or other proteinsolution which it is desired to test for the presence of the homologueof the protein attached to the particle. In order to avoid inhibition ofthe particle coupling by an excess of the homologue, several dilutionsof the solution being tested should be prepared, progressive dilutionsbeing used until it is certain that there are no more than one or a veryfew molecules of homologue for each particle in the suspension. Themixture, with gentle shaking, is held at about 35C for a few minutes toa few hours and is then examined for evidence of particle clumping.Because of the tendency of acrylamide particles to adhere to glass, theacrylamide suspension should be handled in test tubes of polystyrene, orpoly-carbonate plastic, or tubes made of similar non-polar material.(Tubes having acrylamide interior surface would be ideal, but no suchtubes are currently known, by me, to be on the market.) It may be founddesirable to add a small quantity, 0.1 or less, of a surface-activematerial such as Brij 35 (Calbiochem catalog No. 203711), Tween, Dreftor the like to decrease the probability of non-specific clumping andadherence to container surfaces.

Clumping may be detected in one of several ways:

The suspension may be spread on a glass slide or dropped on a piece ofwhite filter paper, or one of the special papers devised for the purpose(R. W. Terry, US. Pat. No. 2,301,717). This suffices to detect grossclumping visually.

Greater sensitivity can be obtained by spreading the suspension afterreaction on a glass microscope slide and observing particle clumpingvisually with a microscope. This is facilitated, in the case offluorescent tags, by ultraviolet excitation and ultraviolet illuminationusing ultraviolet transmitting glass slides and appropriate wavelengthfilters to discriminate against nonfluorescent scattering in the case ofparticles stained with fluorescent dye.

If a radioactive tracer has been used, the paper or slide may be used toprepare an autoradiograph on photographic film, by well-knowntechniques.

In a preferred method, the suspension after reaction is diluted andpassed through a flow cell in a spectrophotofluorometer. This method maybe implemented for best sensitivity and most rapid observation by use ofan apparatus in which the suspension is exposed to a laser beam ofwavelength appropriate to excite the dye, and monitoredphotoelectrically. The particles must be of substantially uniform size,for most applications, and the suspension should be so diluted that twoindividual particles or two particle pairs are rarely present in thecell at one time; in fact, this means that the cell should be void ofparticles about nine-tenths of the time or more. With these conditions,a suitable circuit in the photometer can distinquish between individualand paired particles in the cell. The corresponding events can beindividually counted.

in this way, sensitivity can be so raised that concentrations of only afew hundred molecules in the homologue solution can be detected.

An additional advantage of the fluorescent immuno detection method isthat it is easily carried out automatically.

In another desirable embodiment, the immunoagglomerate after reaction isphysically separated from the fluid and any other constituents of thesuspension, as for example be settling, centrifugation or filtration;the separated agglomerate is washed, and then dissolved (or melted, asfor agar carrier), and the resulting fluid assayed for the fluorescentor other tag. Cellulose acetate carrier, for example, may be dissolvedin acetone; acrylamide or other polymeric carrier may be made withcross-linkages which are amenable to subse quent selective severance.These embodiments are desirable when continuous-fluid-monitoringinstrumentation is preferable to particle-discriminatinginstrumentation.

I claim:

1. The process of forming a reagent, that includes:

a. providing multiple water suspendible polymeric particles, andlabelling each particle generally throughout the volume with amultiplicity of tracer molecules, and

b. covalently bonding to each particle a multiplicity of molecules of asubstance selected from the group consisting of antibodies, antigens andportions and equivalents thereof, by contacting the particles with saidmolecules in aqueous solution in which the particles are dispersed.

2. The reagent formed by the process of claim 1.

3. The process of claim 1 wherein the particles are porous to the tracermolecules.

4. The process that employs the reagent of claim 1 in assaying anaqueous sample for a particular reactant selected from the groupconsisting of antibodies, antigens, portions and equivalents thereof,the substance being the immunological homologue to the reactant, thatincludes the steps c. mixing the reagent with the aqueous sample toproduce linking of multiple particles with units of reactant, thereby toform agglomerate, and

d. detecting tracer concentrations in the agglomerate to therebydetermine the amount of reactant in the sample.

5. The process that employs the reagent of claim 1 in assaying anaqueous sample for a particular reactant selected from the groupconsisting of antibodies, antigens, portions and equivalents thereof,the substance being the immunological homologue to the reactant, thatincludes the steps c. mixing the reagent with the aqueous sample toproduce linking of multiple particles with units of reactant, thereby toform agglomerate, and d. detecting tracer concentrations in anonagglomerated portion of the mix.

6. The process of claim 1 wherein the tracer molecules are selected fromthe group consisting of radioactive and fluorescent molecules.

7. The process of claim 1 wherein said particles are selected from thegroup consisting of polymers of acrylic acid, acrylamide, other acrylicacid derivatives, polymers of styrene and derivatives thereof, agar,agarose, and cellulose acetate.

8. The agglomerate formed by the process that employs the reagent ofclaim 1 in assaying an aqueous sample for a particular reactant selectedfrom the group consisting of antibodies and antigens, the substancebeing the immunological homologue to the reactant, and that includes thesteps of mixing the reagent with the aqueous sample, and continuing saidmixing to produce linking of multiple particles with each unit ofreactant, thereby to form the agglomerate.

1. THE PROCESS OF FORMING A REAGENT, THAT INCLUDES: A. PROVIDING MULTIPLE WATER SUSPENDIBLE POLYMERIC PARTICLES, AND LABELLING EACH PARTICLE GENERALLY THROUGHOUT THE VOLUME WITH A MULTIPLICITY OF TRACER MOLECULES, AND B. COVALENTLY BONDING TO EACH PARTICLE A MULTIPLICITY OF MOLECULES OF A SUBSTANCE SELECTED FROM THE GROUP CONSISTING OF ANTIBODIES, ANTIGENS AND PORTIONS AND EQUIVALENTS THEREOF, BY CONTACTING THE PARTICLES WITH SAID MOLECULES IN AQUEOUS SOLUTION IN WHICH THE PARTICLES ARE DISPERSED.
 2. The reagent formed by the process of claim
 1. 3. The process of claim 1 wherein the particles are porous to the tracer molecules.
 4. The process that employs the reagent of claim 1 in assaying an aqueous sample for a particular reactant selected from the group consisting of antibodies, antigens, portions and equivalents thereof, the substance being the immunological homologue to the reactant, that includes the steps c. mixing the reagent with the aqueous sample to produce linking of multiple particles with units of reactant, thereby to form agglomerate, and d. detecting tracer concentrations in the agglomerate to thereby determine the amount of reactant in the sample.
 5. The process that employs the rEagent of claim 1 in assaying an aqueous sample for a particular reactant selected from the group consisting of antibodies, antigens, portions and equivalents thereof, the substance being the immunological homologue to the reactant, that includes the steps c. mixing the reagent with the aqueous sample to produce linking of multiple particles with units of reactant, thereby to form agglomerate, and d. detecting tracer concentrations in a non-agglomerated portion of the mix.
 6. The process of claim 1 wherein the tracer molecules are selected from the group consisting of radioactive and fluorescent molecules.
 7. The process of claim 1 wherein said particles are selected from the group consisting of polymers of acrylic acid, acrylamide, other acrylic acid derivatives, polymers of styrene and derivatives thereof, agar, agarose, and cellulose acetate.
 8. The agglomerate formed by the process that employs the reagent of claim 1 in assaying an aqueous sample for a particular reactant selected from the group consisting of antibodies and antigens, the substance being the immunological homologue to the reactant, and that includes the steps of mixing the reagent with the aqueous sample, and continuing said mixing to produce linking of multiple particles with each unit of reactant, thereby to form the agglomerate. 